The development of clinically effective cancer vaccines requires reliable analytical methods to evaluate the immune response to vaccination. Immunoassays are among the most sensitive and specific analytical methods that have been used widely in cancer vaccine research, most commonly to monitor the release of cytokines from T-cells in response to vaccination. A typical immunoassay done in a microtiter plate or antibody microarrays, requires 1-5 hours for just the incubation step (during which the binding processes occur) depending on the actual assay. The total reaction time can be much longer depending on the degree of automation. In a research setting where expensive robotic systems are not affordable, a skilled technician performs several key steps in an immunoassay and the technician time is typically more than half the assay cost. This is particularly true when samples cannot be batched for analysis and require immediate attention. A longer assay time therefore adds to the cost of the assay. Human error due to repeated manual intervention is also a major source of inter-assay and intra-assay variation. The emerging concept of a lab-on-a-chip is uniquely positioned to address these issues, by reducing analysis times and providing high levels of automation to increase the productivity in a lab. Due to the limitations in versatility and functionality in the state-of-the-art microchannel-based lab-on-a-chips, we developed an alternative paradigm for microfluidic manipulation based on electrowetting-actuated nanodroplets. In this phase I proposal we will evaluate the feasibility of fully-automated heterogeneous immunoassays on our lab-on-a-chip platform, on nanoliters of sample. We will develop a sandwich ELISA protocol and test it by assaying for cytokine in clinical samples. In phase II, we will optimize the ELISA protocol and integrate multiple immunoassays and process cells on the same platform. Our eventual goal is to develop a nanoliter, rapid, fully automated, hand-held point-of-analysis/point-of-care analyzer that integrates clinical chemistry, molecular diagnostics, and immunoassays. [unreadable] [unreadable] [unreadable]